NEW INSIGHTS INTO THE REGULATION OF MITOCHONDRIAL OUTER MEMBRANE PERMEABILIZATION DURING APOPTOSIS BY Mary Elizabeth Shawgo Submitted to the graduate degree program in Pharmacology, Toxicology & Therapeutics and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Dissertation Committee Members ___________________________ John D. Robertson, Ph.D. (Chair) ___________________________ Dianne Durham, Ph.D. ___________________________ Thomas J. Imig, Ph.D. ___________________________ Hartmut Jaeschke, Ph.D. ___________________________ Gregory A. Reed, Ph.D. ___________________________ Bao-Ting Zhu, Ph.D. Date Defended: August 13, 2009 The Dissertation Committee for Mary Shawgo certifies that this is the approved version of the following dissertation: NEW INSIGHTS INTO THE REGULATION OF MITOCHONDRIAL OUTER MEMBRANE PERMEABILIZATION DURING APOPTOSIS ________________________________ Chairperson - John D. Robertson, Ph.D. Committee: Dianne Durham, Ph.D. Thomas J. Imig, Ph.D. Hartmut Jaeschke, Ph.D. Gregory A. Reed, Ph.D. Bao-Ting Zhu, Ph.D. Date approved: August 13, 2009 ii ACKNOWLEDGEMENTS I feel very fortunate and grateful for all the individuals throughout my education that have encouraged, guided, mentored, loved and supported me from elementary school through graduate school. It has been a long rigorous, frustrating and wearisome journey. I want to start with my family who endured my many years of higher education. I am exceptionally appreciative of my loving parents, Bob and Becky Shawgo; grandparents, Barbara and Stubbie Thorson, Bud and Betty Shawgo; and my brother, and his wife, Thomas and Jenny Shawgo. I would also like to thank my extended family that includes all my uncles, aunts (including greats) and cousins (1st, 2nd, and 3rd). These include but are not limited to my Cousin Mary, Uncle Russ, Dana, Ashley S. and Catherine. I would like to express gratitude to my family, for both the emotional and financial support that were required for me to reach my goal. Thank you for always understanding that school needed to come first for me to succeed. I would like to thank my parents for countless reasons, too many to describe here. I could have never asked for better parents. Thank you for always being my biggest fans and greatest supporters. I am grateful for my Mom for pushing my elementary school teachers. I was ultimately diagnosed with learning disability and found help with school. Without early intervention, this would never have been possible. This allowed me to enjoy the learning process instead of dreading every printed word. I would like to thank my Dad, iii for the hard-work ethic he instilled in me. I would like to thank him for the never-ending proofreading he did on every application for admittance to schools, internships and my new job. Mom and Dad, thank you for being great role models in both work and family. I need to thank some of my learning disability mentors. Thank you, Mrs. Avis Paige from Orchard Lake Elementary School. She was my first learning disability teacher who gave me the skill foundation to compensate for my learning disability and the encouragement to overcome it. Mrs. Donna Watier was my high school disability mentor who opened up the doors to higher education for me and taught me perseverance. Additionally, I need to express lots of gratitude to my educational support at KUMED, Alice Carrott, Brigitte Hofmeister and Carol Wagner. Especially, Alice-she helped me through some of the darkest parts of the woods. During the foremost years of my education I had many teachers who supported me; Mrs. Claudia Nelson, Mrs. Karen Miller and Mr. Tim King. I especially want to thank my fifth grade teacher, Mrs. Miller, who sparked my interest in becoming a scientist. Before this, I did not enjoy school because it was extremely difficult for me. Mrs. Miller changed all of that. She motivated me by teaching science through the use of experiments. Through her hands on approach, I fell in love with learning and, more, specifically science. Additionally, I would like to express thanks for my supporters from Northern Michigan University; Drs. Jackie Bird, William Knox, Ronald Parejko, Lesley iv Putman, Kathleen Thompson, Suzanne Williams, Robert Winn, and Frank Verley. I would especially like to thank Dr. Verley who always gave me the best pep talks. He offered me continued support, encouragement and opportunities which allowed me steady success. Thank you, Dr. Verley, I deeply appreciate everything you have done to help me fulfill my aspirations! I would also like to thank my friends who have supported and encouraged me along the way: Brian Aurand, Theresa & Tom Fisher, Amy Frankson, Sandy Herland, Jessica Lapp, Kara MacNeil, Dr. Michelle Merrihew, Suzy Metzler, Erin Pease, Don Shaffer, and Nagib Ward. Suzy, thanks for proofreading all my non-science papers in undergrad. Last, but not least, I would like to thank all my colleagues at University of Kansas Medical Center. Thank you to all my fellow graduate students. Without the support of fellow graduate students going through the same program, I could not have gotten through graduate school. Thank you, Gina Doss, Colleen Flynn, Dr. Beth Hontz, Erik Pacyniak, Megan Roth, Chieko Saito, Laurie Shannon, Shary Shelton, Ann Thomas, Yi Weaver, and Dr. Rachel Williams. A sincere thank you for making yourselves available to me when I needed a friend or lunch buddy. I would especially like to thank Shary who was the “very best bench mate”. Thanks “Little Dude”. I am glad to have had the opportunity to learn and work with you, Shary. I would like to thank the Pharm/Tox office staff, especially Rosa Meagher, for taking care of all the “behind the scene” tasks that most of the graduate students have no v idea go on. I would like to thank all the Pharm/Tox faculty members for their guidance, knowledge, support and education. I especially would like to thank Dr. Thomas Pazdernik who goes beyond the call of duty in teaching. I would like to thank my dissertation committee members: Drs. Dianne Durham, Hartmut Jaeschke, Gregory Reed, and Bao-Ting Zhu. I am grateful to you for all your contributions. Finally, I would like to thank my mentor, Dr. John Robertson. Thank you for allowing me to join your laboratory and sharing with me your extensive knowledge on apoptosis and cancer chemotherapies. I learned so much! Being a part of your team has been an enriching experience and has made me value different styles. I appreciate the experiences you extended to me in your laboratory. A big heartfelt thanks to all ~ Mary vi ABSTRACT Disruption in normal apoptosis can contribute to the onset of different disease states, including cancer. Additionally, many clinically relevant cancer drugs are effective for their ability to initiate the apoptotic process. Most often this involves the activation the mitochondria-mediated pathway, but the development of specific agents that can activate the receptor-mediated pathway is of increasing interest. Within the existing paradigm of mitochondria-mediated apoptosis, which is activated by intracellular stress, such as DNA damage and cytoskeletal disruption, the pathway is thought to proceed in a linear fashion. Mitochondria outer membrane permeabilization (MOMP) and the release of intermembrane space proteins, such as cytochrome c, are early events during mitochondria-mediated apoptotic signaling. In addition, Apaf-1 is a critical component of the mitochondrial pathway and is generally thought to reside downstream of MOMP. My dissertation investigates the molecular requirements essential for MOMP during stress-induced and receptor-mediated apoptosis. To do so, I used distinct clones of Jurkat T-lymphocytes in which the mitochondria- mediated pathway had been inhibited at three different steps. First, cells stably overexpressed Bcl-2/Bcl-xL to inhibit the activation of Bak, a key regulator of MOMP. Second, cells were stably depleted of Apaf-1 to inhibit the activation of the initiator death protease caspase-9. Finally, cells were stably overexpressed with full-length X-linked inhibitor of apoptosis protein vii (XIAP) to indirectly inhibit activated death proteases caspase-9, -7 and -3. Cells were stably overexpressed with BIR1/BIR2 domains of XIAP to indirectly inhibit activated effector death proteases caspase-3 and -7. The first aim investigated the molecular requirements necessary for Bak activation. Apaf-1-deficient cells and cells overexpressing full-length XIAP or the BIR1/BIR2 domains of XIAP inhibited mitochondrial apoptotic events. The data obtained suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway. As mentioned previously, a second apoptotic pathway is called the receptor-mediated pathway. In some cell types (type II), the mitochondria- mediated pathway is also required for cell death upon stimulation of the receptor-mediated pathway. In the second aim, I used type II Jurkat clones in which the mitochondrial apoptotic pathway had been inhibited at the different steps to investigate the molecular requirements necessary for Fas-mediated apoptosis. Apaf-1-deficient type II Jurkat cells were sensitive to anti-Fas. Inhibiting downstream caspase activation by overexpression of the BIR1/BIR2 domains of XIAP decreased all anti-Fas-induced apoptotic changes. Combined, my findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis does not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells. viii In the third aim, mitochondrial apoptotic events were examined after prolonged treatment with etoposide (> 6 h). Although Apaf-1-deficient cells were resistant to etoposide, total cellular cytochrome c and Smac were decreased after 24 h of incubation. The Apaf-1-deficient cells displayed subtle biochemical markers of autophagy. Interestingly, inhibition of the 26S proteasome by co-treatment of Apaf-1-deficient cells with bortezomib or MG132 led to the robust retention of total cellular cytochrome c and Smac.